Chemotherapy of fungal infections is one of the most difficult and not yet successfully solved problems in modern medicine. This is a consequence of the fact that both, pathogenic fungal organisms, and the humans are eukaryotic organisms and this is the reason of essential difficulties in designing of the selectively acting drugs with low toxicity for the patient. This difficulty has been omitted only in the treatment of topical and intestinal fungal infections. This includes such areas of clinical mycology as gynecology, dermatology, gastroenterology, pulmomology, urology and ophthalmology, where the problem of compounds toxicity occurs to be less dramatic (C. P. Schaffner, in Macrolide antibiotics, S. Omura (red.), Academic Press. Inc., Orlando, p. 457, 1984). The most popular drugs of polyene macrolides used in such cases are Amphotericin B, Nystatin and Pimaricin, which due to the lack of resorption in local and oral administration are practically non-toxic. However, invasive mycoses concerning the infection of internal organs and fungemia are still problems far from successful solution. Current epidemiological statistics concerning the mortality in such types of illnesses are not satisfactory (M. A. Pfaller, D. J. Diekem, Clin. Microbiol. Rev. 20, 133, 2007; T. F. Patterson, Lancet 366, 1013, 2005; S. K Fridkin, Clin. Infect. Dis. 41, 240, 2005). Especially dangerous are invasive candidoses and aspergilloses and infections caused by certain others fungal pathogenes. In the case of invasive candidosis the mortality is in the range 30-70%, aspergillosis more than 50%, the frequency of invasive mycoses in oncology/hematology is approximately 50%, in the case of mycoses of children with leukemia is 29-39% (S. E. Soloviera et al., Rus. Chem. Rev. 80, 103, 2011: A. L. Demain, S. Sanchez, J. Antibiot., 62, 5, 2009; G. O. Bronin et al., Pediatryia 4, 31, 2004). Over 90% of HIV-positive patients suffer from mycoses, and pneumonia caused by Pneumocystis carinii which is the main reason of death in patients with AIDS. Systemic mycoses are common reason of death in adult patients with leukemia. Candida spp., in regard to frequency of incidences, are fourth etiological factor of hospital infections and is a cause of 8-11% of all systemic infections with mortality up to 40%. The frequency of fungal infections in patients after organ transplants is in the range 5-40% depending on the type of transplanted organ. Invasive aspergillosis of lungs is a main reason of patient death after transplant of bone marrow. Blastomycosis, histoplasmosis and coccidiomycosis are endemic mycoses with high frequency of appearance in many regions of the world.
The unfavorable situation in clinical mycology for over 20 years constantly is getting worse for several reasons. One of them is steady increase of infections caused by fungal species previously being non-pathogenic (D. A. Enoch et al., J. Med. Microbiol. 55, 809, 2006; N. Nucii, K. A. Man, Clin. Infect Dis. 41, 521, 2005). The increase of fungal infections is also caused by the use of antibacterial chemotherapeutical agents with broad spectrum and by the use of steroids, and above all by decreasing the immune system activity in increasing number of patients as a consequence of the development of transplantology who required the use of immunosuppressive drugs, and also with the increase of cancer cases and thus usage of immunosuppressive cytostatics (N. Siugh, Med. Mycol. 43, suppl. 1, 267, 2005; A. L. Demain, S. Sanchez, J. Antibiot. 62, 5, 2009).
Especially worrying is the steady decrease of utility of currently clinically available antifungal chemotherapeutics used in the treatment of systemic infections. It is the result of rapid development of resistance of pathogenic fungal strains, above all the multidrug resistant (MDR) ones. The latter phenomenon is a consequence of overexpression of membrane transporter proteins of ABC and MFS superfamilies exporting from the microbial cells xenobiotics as antifungal chemotherapeutic agents (D. Sanglard, Curr. Opinion Microbiol. 5, 378, 2002; M. B. Frosco, J. F. Barrett, Exp. Opin. Invest. Drugs 7, 175, 1998; D. P. Kontoyiannis, R. E. Lewis, Lancet 359, 1135, 2002).
5-fluorocytosine, often used in combination with Amphotericin B to increase its uptake by membrane permeabilisation, as an antimetabolite avoids the exporting activity of MDR transporting proteins, but enhances the development of specific type of resistance mainly by the loss of cytosine permease and cytosine deaminase, which generates in cells the active metabolite—5-fluorouracil. Particularly clinically valuable fungicides of “azoles” group, mostly triazoles, such as myconazol, voriconazol, posaconazol and others, are partially susceptible to their removal from the cells by MDR exporting proteins (R. Franz et al., Antimicrob. Ag. Chemother., 42, 3065, 1998; R. Wakiec et al., Mycoses, 50, 109, 2007). However, being the inhibitors of lanosterol demethylase, interacting with the enzyme, induces changes in the structure of enzymatic protein leading to the loss of inhibitory activity of these compounds. Very valuable and very promising fungicide caspofungin, although with narrow antifungal spectrum but with excellent selectivity, as inhibitor of β-D-glucan synthase interacts with the enzyme, which unfortunately leads to the induction of changes in the structure of the enzyme protein and in consequence the loss of inhibitory activity of the compounds. The reports on growing resistance to the action of this drug start to be published. Therefore, Amphotericin B (fungizone)—from polyene macrolides group practically remains the only one systemic fungicide, which does not induce the development of resistant strains, and not being the substrate of MDR exporting proteins, retains full activity against multidrug resistant strains (M. Ślisz i in., J Antibiot. 60, 436, 2007). Although there are data on the appearance of strains with reduced sensitivity to this antibiotic, as a result of certain changes in the lipid composition of cytoplasmic membrane, but these changes are phenotypic and regressing after discontinued contact with the drug. Moreover, Amphotericin B fulfills also other important requirements for a good antifungal chemotherapeutic such as high activity, broad antifungal spectrum and fungicidal action.
The present situation in clinical mycology points to the necessity for further searches of antifungal drugs. One of the intensively developing research projects concerned study on the modifications of Amphotericin B, aimed at removing of its main shortcomings which are high toxicity and lack of water solubility. However, until now none of the products of antibiotic modification have been introduced to clinical practice. The only practical progress in this area was the introduction to clinical use of Amphotericin B complexes with lipids or liposomal formulations as Abalcet®, Amphotec® and AmBisome®. These formulations of Amphotericin B are however only a little less toxic in comparison to the native antibiotic.
Earlier known derivatives of Amphotericin B are compounds modified mainly at amino group of mycosamine moiety and at carboxyl group of aglycone. The attempts to modify compounds by genetic manipulations of antibiotic producing organism have been also performed. These modifications have been intended to improve the solubility and to reduce the toxicity of the compound. Some of the obtained derivatives had better water solubility due to the introduction of hydrophilic substituents, or by the introduction to the molecules of moieties defining the ionic character of compounds which allows soluble salts to form. However, no significant progress has been achieved in improving the selective toxicity of Amphotericin B derivatives, because no convincing molecular background for the rational modifications has been proposed. The syntheses of derivatives had accidental character and were rather based on random screening.
The prior art on Amphotericin B derivatives includes: 1) derivatives at amino group, 2) derivatives at carboxyl group, 3) double derivatives including the modification of both amino and carboxyl groups, 4) derivatives with genetically modified aglycone fragment. Summary of the current state of knowledge on this matter is presented in the published scientific reviews (A. A. Volmer et al., Nat. Prod. Rep 27, 1329, 2010; S. E. Solovieva et al., Russian Chemical Reviews 80, 103, 2011). In the below mentioned survey of prior art the patent literature is also presented.
The first obtained are Amphotericin B derivatives at amino group. These are N-acyl derivatives (U.S. Pat. No. 3,244,590). An important improvement in this group of compounds deals with N-aminoacyl derivatives of high biological antifungal activity (J. K. Wright et al., J. Antibiol. 35, 911, 1982). There are also known their N,N-dialkylaminoacyl analogs more advantageous as concerns the method of their synthesis (PL 14847). Numerous further compounds were obtained in the group of derivatives at amino and carboxyl functions. A major advancement in the antibiotic modification was the use of N-alkylation reaction. N,N,N-trimethylammonium derivatives (U.S. Pat. No. 4,144,328; Polish Patent 122884), N-alkyl derivatives being products of Michael's addition (A. Czerwinski et al., J. Antibiot. 44, 979, 1991), derivatives being the products of Amadori rearrangement (Polish Patent 82224; U.S. Pat. No. 4,195,172) together with further modifications of glycosyl moiety (U.S. Pat. No. 5,314,999; L. Saint-Julien et al., Antimicrob. Agents Chemother. 36, 2722, 1992) have been obtained. This type of derivatives were further modified as derivatives at amino and carboxyl groups jointly. Major progress in the synthesis of Amphotericin B N-alkyl derivatives was the application of reductive amination reaction with the use of appropriate aldehydes (V. Paquet, E. M. Carreira, Organic Letters 8, 1807, 2006; Europ. Patent Application. EP 1987049A1; International Patent Application WO 2007096137A1; US Patent Application 2009/0186838A1). Also other derivatives, less interesting as regards their properties, such as guanidine derivative (U.S. Pat. No. 4,396,619) and amidine and enamine derivatives (Polish Patent 120111) have been obtained.
Much fewer derivatives at carboxyl group have been obtained. The first compound of this type which caused a great interest was methyl ester of Amphotericin B (U.S. Pat. No. 4,035,567) and a number of its water soluble salts (U.S. Pat. Nos. 3,914,409; 6,613,889B2; 4,041,232; Patent Application PCT WO 2007/06335A2). Also other esters of the antibiotic and their salts were obtained (U.S. Pat. No. 5,981,729; S. Stefanska et al., Acta Polon. Pharm. 40, 1, 1983), other derivatives at carboxyl group comprised hydrazides (K. Grzybowska, E. Borowski, J. Antibiot. 43, 907, 1990), (PL 122086; PL 199213) and their water soluble salts (Polish Patent 138831).
A large group of Amphotericin B derivatives are compounds in which a number of the above mentioned substituents at amino and carboxyl groups were combined in one compound. There are amides and esters of N,N-dialkyl derivatives (WO 2009/0186838A1; WO 2007/096137A1), esters and amides of glycosyl derivatives and their N-alkyl derivatives (U.S. Pat. Nos. 6,562,796B2; 6,664,241B2), amides and esters of N-alkyl and N-aminoacyl derivatives (PL 199213), methyl esters of N-alkyl N-glycosyl derivatives (PL 180253), esters of guanidine derivatives (U.S. Pat. No. 4,396,610), methyl ester of N-amino and amidine derivatives (PL 120035), esters and amides of dialkylaminoacyl derivative (PL 142848).
Separate group of products of Amphotericin B modifications constitute compounds and their various derivatives with modified macrolide part of the antibiotic molecule. These compounds are not relevant to the subject matter of the present invention, which relates to Amphotericin B derivatives with unmodified macrolide ring, but for the sake of the complete information on prior art, relevant patents and patent applications are mentioned. Beecham group documents include: (U.S. Pat. Nos. 6,284,736; 5,116,960; 5,066,646; 5,100,876; EP 0350164; WO 91/09047; EP 0431870; EP 0375222; EP 0431870. Smith-Kline Beecham group documents include: WO 93/16090; WO 93/14100; WO 93/17034.
Above presented the state of prior art allows to put forward the following conclusion. In spite of a large experimental data and a very large number of Amphotericin B derivatives obtained, none of these compounds have yet entered the stage of advanced clinical trials and industrial development, because the essential improvement of their properties in relation to the native antibiotic was not achieved.
The background of the invention is the new idea which enables the Amphotericin B modification, aimed to obtain the most desired effect which is essential increase of selective toxicity of antibiotic derivatives. Our study has shown that selective toxicity of Amphotericin B derivatives is only to the limited extent the result of differential affinity of derivatives to their molecular targets in fungal (ergosterol) and mammalian (cholesterol) cells indispensable for the creation of lethal channels (M. Baginski et al., Bichim. Biophys. Acta 1567, 223, 2002). Differences of that affinity cannot by essentially increased by the chemical modification of the antibiotic and thus some changes in the affinity to both molecular targets can give rather limited increase of selective toxicity effect of the modified compounds. We suggest that the derivatives of Amphotericin B with somewhat modified differential affinity to cholesterol and ergosterol, should be named derivatives of the first generation. Much more possibility for the essential increase of selective toxicity of Amphotericin B derivatives gives, observed by us, phenomenon of differential ability to create lethal channels in fungal and mammalian cells not only as a result of differences of compounds affinity to molecular targets in both types of organisms but above all as a result of differential ability of formed antibiotic—molecular target complex to aggregate leading to the formation of a lethal membrane channels. This occurs in the case of N-substituted antibiotic derivatives containing voluminous or bulky moieties which can induce steric hindrance effect (J. Szlinder-Rychert et al., Biochem. Biophys. Acta 1528, 15, 2001; J. Szlinder-Rychert et al., Il Farmaco 59, 289, 2004). It is possible, that Amphotericin B derivatives with bulky substituents at amino group, form antibiotic-sterol complexes with modified geometry and thus result in the differential ability to aggregate into lethal membrane channels with ergosterol and cholesterol containing membranes. It is also important, that spatially hindered compounds retain their high activity towards fungal strains with multidrug resistance (M. Slisz et al., J. Antibiot. 60, 436, 2007). Such type of antibiotic derivatives we call the derivatives of second generation. In contrast to the derivatives at amino group, the spatially hindered derivatives at carboxyl group do not give the essential improvement of selectivity effect. The basic derivatives such as esters and amides can only augment the advantageous properties of spatially hindrance N-derivatives, facilitating the formation of soluble salts with acids. Amphoteric derivatives can form soluble salts with bases, because bulky N-substituents break the zwitterionic structure of the native antibiotic.
The beneficial effect for the increase of selective toxicity of the introduction of steric hindrance at amino group of mycosamine moiety of the antibiotic molecule is achieved, providing that the basic character of amino group is preserved (N-alkyl derivatives), or as a new amino group is present in the substituent (N-aminoacyl derivatives). The particularly important role of this group in the interaction with sterole molecular targets has been evidenced (M. Baginski et al., Biophys. Chem. 49, 241, 1994). Discussed above idea on the positive effect of spatially hindered N-substituted Amphotericin B derivatives does not allow to identify the exact molecular structures which should have the compounds with optimal properties. Synthesis and selection of the most favorable compounds is still a matter of empiricism. The first group of such derivatives was obtained (PL 210774). However further empirical studies were needed for the identification of the most favorable compounds. This aim has been achieved within the present invention, as a result of unexpected identification of sterically hindered derivatives with most advantageous selectivity.
The advantage of new semisynthetic Amphotericin B derivatives, according to the invention, is that they exhibit high antifungal activity against a broad spectrum of microorganism of the Candida species and filamentous fungi, as well as towards multidrug resistant (MDR) strains with overexpression of transporter protein Cdr1p and Cdr2p. These derivatives also exhibit low hemotoxicity, which is essential factor of toxicity of polyene macrolides, and form water soluble salts.
Unexpected novelty permitting to obtain, according to the invention, advantageous effect of hemotoxicity reduction, is introduction to the substituents at amino group of Amphotericin B of new appropriate bulky moieties, which may induce the steric hindrance effect. It appeared that such steric hindrance factor decreases lethal permeabilising activity of Amphotericin B derivatives at greater degree towards mammalian than fungal cells, what increases their selectivity of action and essentially reduces hemotoxity of these compounds. Bulky moieties which may give the optimal effect of steric hindrance include ring systems carbo-, as well as heterocyclic, alicyclic and aromatic, bulky substituents as tert-butyl, nitro group, bromine atom and also aliphatic moieties which due to their flexibility can form voluminous conformational structures.